The present invention provides novel compounds, novel compositions and methods for their use and manufacture. The compounds and compositions of the present invention are generally useful pharmacologically as therapeutic agents in disease states alleviated by the inhibition or antagonism of protein kinase activated signalling pathways in general, and in particular in the pathological processes which involve aberrant cellular proliferation, such disease states including tumor growth, restenosis, atherosclerosis, and thrombosis. In particular, the present invention relates to a series of substituted aza-oxindole compounds, which exhibit protein tyrosine kinase and protein serine/threonine kinase inhibition, and which are useful for the prevention of chemotherapy-induced alopecia.
Protein kinases play a critical role in the control of cell growth and differentiation and are key mediators of cellular signals leading to the production of growth factors and cytokines. See, for example, Schlessinger and Ullrich, Neuron 1992, 9, 383. A partial, non-limiting, list of such kinases includes abl, ARaf, ATK, ATM, bcr-abl, BIk, BRaf, Brk, Btk, CDK1, CDK2, CDK3, CDK4, CDK5, CDK6, CDK7, CDK8, CDK9, cfms, c-fms, c-kit, c-met, cRaf1, CSF1R, CSK, c-src, EGFR, ErbB2, ErbB3, ErbB4, ERK, ERK1, ERK2, Fak, fes, FGFR1, FGFR2, FGFR3, FGFR4, FGFR5, Fgr, FLK-4, Fps, Frk, Fyn, GSK, gsk3a, gsk3b, Hck, IGF-1R, IKK, IKK1, IKK2, IKK3, INS-R, Integrin-linkedkinase, Jak, JAK1, JAK2, JAK3, JNK, JNK, Lck, Lyn, MEK, MEK1, MEK2, p38, PDGFR, PIK, PKB1, PKB2, PKB3, PKC, PKCxcex1, PKCxcex2, PKCxcex4, PKCxcex5, PKCxcex3, PKCxcex, PKCxcexc, PKCxcex6, PLK1, Polo-like kinase, PYK2, tie1, tie2, TrkA, TrkB, TrkC, UL13, UL97, VEGF-R1, VEGF-R2, Yes and Zap70. Protein kinases have been implicated as targets in central nervous system disorders such as Alzheimer""s (Mandelkow, E. M. et al. FEBS Lett. 1992, 314, 315; Sengupta, A. et al. Mol. Cell. Biochem. 1997, 167,99), pain sensation (Yashpal, K. J. Neurosci. 1995, 15, 3263-72), inflammatory disorders such as arthritis (Badger, J. Pharm. Exp. Ther. 1996, 279, 1453), psoriasis (Dvir, et al, J. Cell Biol. 1991, 113, 857), bone diseases such as osteoporosis (Tanaka et al, Nature, 1996, 383, 528), cancer (Hunter and Pines, Cell 1994, 79, 573), atherosclerosis (Hajjar and Pomerantz, FASEB J. 1992, 6, 2933), thrombosis (Salari, FEBS 1990, 263, 104), metabolic disorders such as diabetes (Borthwick, A. C. et al. Biochem. Biophys. Res. Commun. 1995, 210, 738), blood vessel proliferative disorders such as angiogenesis (Strawn et al Cancer Res. 1996, 56, 3540; Jackson et al J. Pharm. Exp. Ther. 1998, 284, 687), restenosis (Buchdunger et al, Proc, Nat. Acad. Sci USA 1991, 92, 2258), autoimmune diseases and transplant rejection (Bolen and Brugge, Ann. Rev. Immunol. 1997, 15, 371) and infectious diseases such as viral (Littler, E. Nature 1992, 358, 160), and fungal infections (Lum, R. T. PCT Int. Appl., WO 9805335 A1 980212).
The signals mediated by kinases have also been shown to control growth, death and differentiation in the cell by regulating the processes of the cell cycle (Massague and Roberts, Current Opinion in Cell Biology 1995, 7, 769-72). Progression through the eukaryotic cell cycle is controlled by a family of kinases called cyclin dependent kinases (CDKs) (Myerson, et al., EMBO Journal 1992, 11, 2909). The coordinate activation and inactivation of different cyclin/CDK complexes is necessary for normal progression through the cell cycle (Pines, Trends in Biochemical Sciences 1993, 18, 195; Sherr, Cell 1993, 73, 1059). Both the critical G1-S and G2-M transitions are controlled by the activation of different cyclin/CDK activities. In G1, both cyclin D/CDK4 and cyclin E/CDK2 are thought to mediate the onset of S-phase (Matsushime, et al., Molecular and Cellular Biology 1994, 14, 2066; Ohtsubo and Roberts, Science 1993, 259, 1908; Quelle, et al., Genes and Development 1993, 7, 1559; Resnitzky, et al., Molecular and Cellular Biology 1994, 14, 1669). Progression through S-phase requires the activity of cyclin A/CDK2 (Girard, et al., Cell 1991, 67, 1169; Pagano, et al., EMBO Journal 1992, 11, 961; Rosenblatt, et al., Proceedings of the National Academy of Science USA 1992, 89, 2824; Walker and Maller, Nature 1991, 354, 314; Zindy, et al., Biochemical and Biophysical Research Communications 1992, 182, 1144) whereas the activation of cyclin A/cdc2 (CDK1) and cyclin B/cdc2 are required for the onset of metaphase (Draetta, Trends in Cell Biology 1993, 3, 287; Murray and Kirschner, Nature 1989, 339, 275; Solomon, et al., Molecular Biology of the Cell. 1992, 3, 13; Girard, et al., Cell 1991, 67, 1169; Pagano, et al., EMBO Joumal 1992, 11, 961; Rosenblatt, et al., Proceedings of the National Academy of Science USA 1992, 89, 2824; Walker and Maller, Nature 1991, 354, 314; Zindy, et al., Biochemical and Biophysical Research Communications 1992, 182, 1144). It is not surprising, therefore, that the loss of control of CDK regulation is a frequent event in hyperproliferative diseases and cancer (Pines, Current Opinion in Cell Biology 1992, 4, 144; Lees, Current Opinion in Cell Biology 1995, 7, 773; Hunter and Pines, Cell 1994, 79, 573). The selective inhibition of CDKs is therefore an object of the present invention.
In brief summary, the invention comprises compounds of the formula (I):

wherein X is selected from the group consisting of: N, CH, CCF3, and C(C1-12 aliphatic);
Y is C or N, with the proviso that when Y is N, R1 is absent, and Z, A and D are each C;
Z is C or N, with the proviso that when Z is N, R2 is absent, and Y, A and D are each C;
A is C or N, with the proviso that when A is N, R3 is absent, and Y, Z and D are each C;
D is C or N, with the proviso that when D is N, then Y, Z and A are each C; with the further proviso that Y, Z, A and D do not simultaneously all represent C;
R1 is selected from the group consisting of: hydrogen, C1-12 aliphatic, thiol, hydroxy, hydroxy-C1-12 aliphatic, Aryl, Aryl-C1-12 aliphatic, R6-Aryl-C1-12 aliphatic, Cyc, Cyc-C1-6 aliphatic, Het, Het-C1-12 aliphatic, C1-12 alkoxy, Aryloxy, amino, C1-12 aliphatic amino, di-C1-12 aliphatic amino, di-C1-12 aliphatic aminocarbonyl, di-C1-12 aliphatic aminosulfonyl, C1-12 alkoxycarbonyl, halogen, cyano, sulfonamide and nitro, where R6, Aryl, Cyc and Het are as defined below;
R2 is selected from the group consisting of: hydrogen, C1-12 aliphatic, N-hydroxyimino-C1-12 aliphatic, C1-12 alkoxy, hydroxy-C1-12 aliphatic, C1-12 alkoxycarbonyl, carboxyl C1-12 aliphatic, Aryl, R6-Aryl-oxycarbonyl, R6-oxycarbonyl-Aryl, Het, aminocarbonyl, C1-12 aliphatic-aminocarbonyl, Aryl-C1-12 aliphatic-aminocarbonyl, R6-Aryl-C1-12 aliphatic-aminocarbonyl, Het-C1-12 aliphatic-aminocarbonyl, hydroxy-C1-12 aliphatic-aminocarbonyl, C1-12-alkoxy-C1-12 aliphatic-aminocarbonyl, C1-12 alkoxy-C1-12 aliphatic-amino, di-C1-12 aliphatic amino, di-C1-12 aliphatic aminocarbonyl, di-C1-12 aliphatic aminosulfonyl, halogen, hydroxy, nitro, C1-12 aliphatic-sulfonyl, aminosulfonyl and C1-12 aliphatic-aminosulfonyl, where R6 Aryl and Het are as defined below;
R1 and R2 are optionally joined to form a fused ring selected from the group as defined for Het below, and said fused ring is optionally substituted by one or more substituents selected from the group consisting of: C1-12 aliphatic, halogen, nitro, cyano, C1-12 alkoxy, carbonyl-C1-12 alkoxy and oxo;
R3 is selected from the group consisting of: hydrogen, C1-12 aliphatic, hydroxy, hydroxy C1-12 aliphatic, di-C1-12 aliphatic amino, di-C1-12 aliphatic aminocarbonyl, di-C1-12 aliphatic aminosulfonyl, C1-12 alkoxy, Aryl, Aryloxy, hydroxy-Aryl, Het, hydroxy-Het, Het-oxy and halogen, where Aryl and Het are as defined below;
R2 and R3 are optionally joined to form a fused ring selected from the group as defined for Het below, and said fused ring is optionally substituted by C1-6 aliphatic and/or C1-6 aliphatic-carbonyl;
R4 is selected from the group consisting of: sulfonic acid, C1-12 aliphatic-sulfonyl, sulfonyl-C1-12 aliphatic, C1-12 aliphatic-sulfonyl-C1-6 aliphatic, C1-6 aliphatic-amino, R7-sulfonyl, R7-sulfonyl-C1-12 aliphatic, R7-aminosulfonyl, R7-aminosulfonyl-C1-12 aliphatic, R7-sulfonylamino, R7-sulfonylamino-C1-12 aliphatic, aminosulfonylamino, di-C1-12 aliphatic amino, di-C1-12 aliphatic aminocarbonyl, di-C1-12 aliphatic aminosulfonyl, di-C1-12 aliphatic amino, di-C1-12 aliphatic aminocarbonyl, di-C1-12 aliphatic aminosulfonyl-C1-12 aliphatic, (R8)1-3-Arylamino, (R8)1-3-Arylsulfonyl, (R8)1-3-Aryl-aminosulfonyl, (R8)1-3-Aryl-sulfonylamino, Het-amino, Het-sulfonyl, Het-aminosulfonyl, aminoiminoamino and aminoiminoaminosulfonyl, where R7, R8, Aryl and Het are as defined below;
R5 is hydrogen or R4 and R5 are optionally joined to form a fused ring selected from the group as defined for Het below, and said fused ring is optionally substituted by one or more substituents selected from the group consisting of: C1-12 aliphatic, oxo and dioxo;
R6 is selected from the group consisting of: C1-12 aliphatic, hydroxy, C1-12 alkoxy and halogen;
R7 is selected from the group consisting of: hydrogen, C1-12 aliphatic, C1-12 alkoxy, hydroxy-C1-12 alkoxy, hydroxy-C1-12 aliphatic, carboxylic acid, C1-12 aliphatic-carbonyl, Het, Het-C1-12-aliphatic, Het-C1-12-alkoxy, di-Het-C1-12-alkoxy Aryl, Aryl-C1-12-aliphatic, Aryl-C1-12-alkoxy, Aryl-carbonyl, C1-18 alkoxyalkoxyalkoxyalkoxyaliphatic and hydroxyl, where Het and Aryl are as defined below;
R8 is selected from the group consisting of: hydrogen, nitro, cyano, C1-12 alkoxy, halo, carbonyl-C1-12 alkoxy and halo-C1-12aliphatic;
Aryl is selected from the group consisting of: phenyl, naphthyl, phenanthryl and anthracenyl;
Cyc is selected from the group consisting of: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl, and optionally has one or more degrees of unsaturation;
Het is a saturated or unsaturated heteroatom ring system selected from the group consisting of: benzimidazole, dihydrothiophene, dioxin, dioxane, dioxolane, dithiane, dithiazine, dithiazole, dithiolane, furan, imidazole, isoquinoline, morpholine, oxazole, oxadiazole, oxathiazole, oxathiazolidine, oxazine, oxadiazine, piperazine, piperadine, pyran, pyrazine, pyrazole, pyridine, pyrimidine, pyrrole, pyrrolidine, quinoline, tetrahydrofuran, tetrazine, thidiazine, thiadiazole, thiatriazole, thiazine, thiazole, thiomorpholine, thiophene, thiopyran, triazine and triazole; and the salts, esters, amides, carbamates, solvates, polymorphs, hydrates, polymorphs, affinity reagents and/or prodrugs thereof, in either crystalline or amorphous form.
The esters, amides and and carbamates are preferably hydrolyzable and are more preferably biohydrolyzable. The salts are preferably pharmaceutically acceptable salts.
A more preferred genus of compounds of the present invention includes compounds of formula (I), defined as follows:

wherein X is selected from the group consisting of: N, CH and C(C1-6 aliphatic);
Y is C or N, with the proviso that when Y is N, R1 is absent, and Z, A and D are each C;
Z is C or N, with the proviso that when Z is N, R2 is absent, and Y, A and D are each C;
A is C or N, with the proviso that when A is N, R3 is absent, and Y, Z and D are each C;
D is C or N, with the proviso that when D is N, then Y, Z and A are C; with the further proviso that Y, Z, A and D do not simultaneously all represent C;
R1 is selected from the group consisting of: hydrogen, C1-6 aliphatic, hydroxy-C1-6 aliphatic, Aryl-C1-6 aliphatic, R6-Aryl-C1-6 aliphatic, Cyc-C1-6 aliphatic, Het-C1-6 aliphatic, C1-6 alkoxy, Aryloxy, aminocarbonyl, di-C1-6 aliphatic amino, di-C1-6 aliphatic aminocarbonyl, di-C1-6 aliphatic aminosulfonyl, C1-6 alkoxycarbonyl, halogen and nitro, where R6, Aryl, Cyc and Het are as defined below;
R2 is selected from the group consisting of: hydrogen, C1-6 aliphatic, R7-C1-6 aliphatic, C1-6 alkoxy, hydroxy-C1-6 aliphatic, C1-6 alkoxycarbonyl, carboxyl C1-6 aliphatic, Aryl, R6-Aryl-oxycarbonyl, R6-oxycarbonyl-Aryl, Het, aminocarbonyl, C1-6 aliphatic-aminocarbonyl, Aryl-C1-6 aliphatic-aminocarbonyl, R6-Aryl-C1-6 aliphatic-aminocarbonyl, Het-C1-6 aliphatic-aminocarbonyl, hydroxy-C1-6 aliphatic-aminocarbonyl, C1-6-alkoxy-C1-6 aliphatic-aminocarbonyl, C1-6 alkoxy-C1-6 aliphatic-amino, di-C1-6 aliphatic amino, di-C1-6 aliphatic aminocarbonyl, di-C1-6 aliphatic aminosulfonyl, halogen, hydroxy, nitro, sulfo, C1-6 aliphatic-sulfonyl, aminosulfonyl, C1-6 aliphatic-aminosulfonyl and quaternary ammonium, where R6, R7, Aryl and Het are as defined below;
R1 and R2 are optionally joined to form a fused ring selected from the group as defined for Het below, and said fused ring is optionally substituted by halogen and/or oxo;
R3 is selected from the group consisting of: hydrogen, C1-6 aliphatic, hydroxy, hydroxy C1-6 aliphatic, di-C1-6 aliphatic amino, di-C1-6 , aliphatic aminocarbonyl, di-C1-6 aliphatic aminosulfonyl, C1-6 alkoxy, Aryl, Aryloxy, hydroxy-Aryl, Het, hydroxy-Het, Het-oxy and halogen, where Aryl and Het are as defined below;
R2 and R3 are optionally joined to form a fused ring selected from the group as defined for Het below, and said fused ring is optionally substituted by C1-6 aliphatic or C1-6 aliphatic-carbonyl;
R4 is selected from the group consisting of: sulfonic acid, C1-12 aliphatic-sulfonyl, sulfonyl-C1-12 aliphatic, C1-12 aliphatic-sulfonyl-C1-6 aliphatic, C1-6 aliphatic-amino, R7-sulfonyl, R7-sulfonyl-C1-12 aliphatic, R7-aminosulfonyl, R7-aminosulfonyl-C1-12 aliphatic, R7-sulfonylamino, R7-sulfonylamino-C1-12 aliphatic, aminosulfonylamino, di-C1-12 aliphatic amino, di-C1-12 aliphatic aminocarbonyl, di-C1-12 aliphatic aminosulfonyl, di-C1-12 aliphatic amino, di-C1-12 aliphatic aminocarbonyl, di-C1-12 aliphatic aminosulfonyl-C1-12 aliphatic, (R8)1-3-Arylamino, (R8)1-3-Arylsulfonyl, (R8)1-3-Aryl-aminosulfonyl, (R8)1-3-Aryl-sulfonylamino, Het-amino, Het-sulfonyl, Het-aminosulfonyl, aminoiminoamino and aminoiminoaminosulfonyl, where R7, R8, Aryl and Het are as defined below;
R5is hydrogen;
R4 and R5 are optionally joined to form a fused ring selected from the group as defined for Het below, and said fused ring is optionally substituted by oxo or dioxo;
R6 is selected from the group consisting of: hydrogen, C1-6 aliphatic, hydroxy, C1-6 alkoxy and halogen;
R7 is selected from the group consisting of: hydrogen, C1-12 aliphatic, C1-12 alkoxy, hydroxy-C1-12 alkoxy, hydroxy-C1-12 aliphatic, carboxylic acid, C1-12 aliphatic-carbonyl, Het, Het-C1-12-aliphatic, Het-C1-12-alkoxy, di-Het-C1-12-alkoxy Aryl, Aryl-C1-12-aliphatic, Aryl-C1-12-alkoxy, Aryl-carbonyl, C1-18 alkoxyalkoxyalkoxyalkoxyaliphatic and hydroxyl, where Het and Aryl are as defined below;
R8 is hydrogen and/or halo-C1-6 aliphatic;
Aryl is phenyl or naphthyl;
Cyc is selected from the group consisting of: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl, and optionally has one or more degrees of unsaturation;
Het is a saturated or unsaturated heteroatom ring system selected from the group consisting of: benzimidazole, dihydrothiophene, dioxin, dioxane, dioxolane, dithiane, dithiazine, dithiazole, dithiolane, furan, imidazole, morpholine, oxazole, oxadiazole, oxathiazole, oxathiazolidine, oxazine, oxadiazine, piperazine, piperadine, pyran, pyrazine, pyrazole, pyridine, pyrimidine, pyrrole, pyrrolidine, tetrahydrofuran, tetrazine, thiadiazine, thiadiazole, thiatriazole, thiazine, thiazole, thiomorpholine, thiophene, thiopyran, triazine and triazole; and
the salts, esters, amides, carbamates, solvates, polymorphs, hydrates, affinity reagents and/or prodrugs thereof, in either crystalline or amorphous form. The esters, amides and carbamates are preferably hydrolyzable and are more preferably biohydryzeable. The salts are preferably pharmaceutically acceptable salts.
A highly preferred genus of compounds of the present invention includes compounds of formula (I), defined as follows:

wherein X is selected from the group consisting of: N, CH and CCH3;
Y is C or N, with the proviso that when Y is N, R1 is absent, and Z, A and D are each C;
Z is C or N, with the proviso that when Z is N, R2 is absent, and Y, A and D are each C;
A is C or N, with the proviso that when A is N, R3 is absent, and X, Y and D are each C;
D is C or N, with the proviso that when D is N, then Y, Z and A are each C; with the further proviso that Y, Z, A and D do not simultaneously all represent C;
R1 is selected from the group consisting of: hydrogen, C1-6 aliphatic, hydroxy-C1-6 aliphatic, di-C1-6 aliphatic amino, di-C1-6 aliphatic aminocarbonyl, di-C1-6 aliphatic aminosulfonyl, Aryl-C1-6 aliphatic, R6-Aryl-C1-6 aliphatic, Cyc-C1-6 aliphatic, Het-C1-6 aliphatic, C1-6 alkoxy, Aryloxy, aminocarbonyl, C1-6 alkoxycarbonyl, halogen and nitro, where R6, Aryl, Cyc and Het are as defined below;
R2 is selected from the group consisting of: hydrogen, C1-6 aliphatic, N-hydroxyimino-C1-6 aliphatic, C1-6 alkoxy, C1-6 alkoxycarbonyl, Aryl, R6-Aryloxycarbonyl, Het, aminocarbonyl, C1-6 aliphatic aminocarbonyl, Aryl-C1-6 aliphatic aminocarbonyl, R6-Aryl-C1-6 aliphatic aminocarbonyl, Het-C1-6 aliphatic aminocarbonyl, di-C1-6 aliphatic amino, di-C1-6 aliphatic aminocarbonyl, di-C1-6 aliphatic aminosulfonyl, hydroxy-C1-6 aliphatic aminocarbonyl, C1-6-alkoxy-C1-6 aliphatic aminocarbonyl, C1-6 alkoxy-C1-6 aliphatic amino, halogen, hydroxy, nitro, C1-6 aliphatic sulfonyl, aminosulfonyl and C1-6 aliphatic aminosulfonyl, where R6, Aryl and Het are as defined below;
R1 and R2 are optionally joined to form a fused ring selected from the group as defined for Het below, and said fused ring is optionally substituted by halogen and/or oxo;
R3 is selected from the group consisting of: hydrogen, C1-6 aliphatic, hydroxy, hydroxy C1-6 aliphatic, di-C1-6 aliphatic amino, di-C1-6 aliphatic aminocarbonyl, di-C1-6 aliphatic aminosulfonyl C1-6 alkoxy, Aryloxy, Het and halogen, where Aryl and Het are as defined below;
R2 and R3 are optionally joined to form a fused ring selected from the group as defined for Het below, and said fused ring is optionally substituted by C1-6 alkyl and/or C1-6 alkylcarbonyl;
R4 is selected from the group consisting of: R7-sulfonyl, R7-sulfonyl C1-6-aliphatic, C1-6 aliphatic sulfonyl-C1-6 aliphatic, R7-aminosulfonyl, di-C1-6 aliphatic amino, di-C1-6 aliphatic aminocarbonyl, di-C1-6 aliphatic aminosulfonyl, di-C1-6 aliphatic aminosulfonyl-C1-6 aliphatic, R7-aminosulfonyl C1-6 aliphatic, aminosulfonylamino, R7-C1-6 aliphatic aminosulfonyl-C1-6 aliphatic, Aryl, Het, R8-Aryl-aminosulfonyl, Het-aminosulfonyl and aminoiminoaminosulfonyl, where R7, R8, Aryl and Het are as defined below;
R5 is hydrogen;
R4 and R5 are optionally joined to form a fused ring selected from the group as defined for Het below, and said used ring is optionally substituted by oxo or dioxo;
R6 is selected from the group consisting of: hydroxy, C1-6 alkoxy and halogen;
R7 is selected from the group consisting of: hydrogen, C1-6 aliphatic, hydroxy C1-6-alkoxy, hydroxy-C1-6 aliphatic, C1-6 aliphatic carbonyl, Aryl-carbonyl, C1-12 alkoxyalkoxyalkoxyalkoxyalkyl, hydroxyl, Aryl, Aryl-C1-6-alkoxy, Aryl-C1-6-aliphatic, Het, Het-C1-6-alkoxy, di-Het-C1-6-alkoxy, Het-C1-6-aliphatic and di-Het-C1-6-aliphatic;
R8 is trifluoromethyl;
Aryl is phenyl;
Cyc is cyclobutyl;
Het is a saturated or unsaturated heteroatom ring system selected from the group consisting of: benzimidazole, dihydrothiophene, dioxolane, furan, imidazole, morpholine, oxazole, pyridine, pyrrole, pyrrolidine, thiadiazole, thiazole, thiophene, and triazole;
and the salts, esters, amides, carbamates, solvates, polymorphs, hydrates, affinity reagents and/or prodrugs thereof, in either crystalline or amorphous form. The esters, amides and carbamates are preferably hydrolyzable and are more preferably biohydrolyzable. The salts are preferably pharmaceutically acceptable salts.
A preferred group of compounds of the present invention with respect to the substitutions at R4 are compounds of formula (I):

wherein X is N or CH;
Y is C or N, with the proviso that when Y is N, R1 is absent, and Z, A and D are each C;
Z is C or N, with the proviso that when Z is N, R2 is absent, and Y, A and D are each C;
A is C or N, with the proviso that when A is N, R3 is absent, and Y, Z and D are each C;
D is C or N, with the proviso that when D is N, Y, Z and A are each C;
with the further proviso that Y, Z, A and D do not simultaneously all represent C;
R1 is selected from the group consisting of: hydrogen, C1-12 aliphatic, thiol, hydroxy, hydroxy-C1-12 aliphatic, Aryl, Aryl-C1-12 aliphatic, R6-Aryl-C1-12 aliphatic, Cyc, Cyc-C1-6 aliphatic, Het, Het-C1-12 aliphatic, C1-12 alkoxy, Aryloxy, amino, C1-12 aliphatic amino, di-C1-12 aliphatic amino, di-C1-12 aliphatic aminocarbonyl, di-C1-12 aliphatic aminosulfonyl, C1-12 alkoxycarbonyl, halogen, cyano, sulfonamide and nitro, where R6, Aryl, Cyc and Het are as defined below;
R2 is selected from the group consisting of: hydrogen, C1-12 aliphatic, N-hydroxyimino-C1-12 aliphatic, C1-12 alkoxy, hydroxy-C1-12 aliphatic, C1-12 alkoxycarbonyl, carboxyl C1-12 aliphatic, Aryl, R6-Aryl-oxycarbonyl, R6-oxycarbonyl-Aryl, Het, aminocarbonyl, C1-12 aliphatic-aminocarbonyl, Aryl-C1-12 aliphatic-aminocarbonyl, R6-Aryl-C1-12 aliphatic-aminocarbonyl, Het-C1-12 aliphatic-aminocarbonyl, hydroxy-C1-12 aliphatic-aminocarbonyl, C1-12-alkoxy-C1-12 aliphatic-aminocarbonyl, C1-12 alkoxy-C1-12 aliphatic-amino, di-C1-12 aliphatic amino, di-C1-12 aliphatic aminocarbonyl, di-C1-12 aliphatic aminosulfonyl, halogen, hydroxy, nitro, C1-12 aliphatic-sulfonyl, aminosulfonyl and C1-12 aliphatic-aminosulfonyl, where R6, Aryl and Het are as defined below;
R1 and R2 are optionally joined to form a fused ring selected from the group as defined for Het below, and said fused ring is optionally substituted by one or more substituents selected from the group consisting of: halogen, nitro, cyano, C1-12 alkoxy, carbonyl-C1-12 alkoxy and oxo;
R3 is selected from the group consisting of: hydrogen, C1-12 aliphatic, hydroxy, hydroxy C1-12 aliphatic, di-C1-12 aliphatic amino, di-C1-12 aliphatic aminocarbonyl, di-C1-12 aliphatic aminosulfonyl, C1-12 alkoxy, Aryl, Aryloxy, hydroxy-Aryl, Het, hydroxy-Het, Het-oxy, or halogen, where Aryl and Het are as defined below;
R2 and R3 are optionally joined to form a fused ring selected from the group as defined for Het below, and said fused ring is optionally substituted by C1-6 aliphatic and/or C1-6 aliphatic-carbonyl;
R4 is selected from the group consisting of: R7-aminosulfonyl, R7-aminosulfonyl-C1-12 aliphatic, R7-sulfonylamino, R7-sulfonylamino-C1-12 aliphatic, aminosulfonylamino, di-C1-12 aliphatic aminosulfonyl, di-C1-12 aliphatic aminosulfonyl-C1-12 aliphatic, (R8)1-3-Aryl-aminosulfonyl, (R8)1-3-Aryl-sulfonylamino and aminoiminoaminosulfonyl, where R7, R8, Aryl and Het are as defined below;
R5is hydrogen;
R4 and R5 are optionally joined to form a fused ring selected from the group as defined for Het below, and said fused ring is optionally substituted by oxo or dioxo;
R6 is selected from the group consisting of: C1-12 aliphatic, hydroxy, C1-12 alkoxy and halogen;
R7 is selected from the group consisting of: hydrogen, C1-12 aliphatic, C1-12 alkoxy, hydroxy-C1-12 alkoxy, hydroxy-C1-12 aliphatic, carboxylic acid, C1-12 aliphatic-carbonyl, Het, Het-C1-12-aliphatic, Het-C1-12-alkoxy, di-Het-C1-12-alkoxy Aryl, Aryl-C1-12-aliphatic, Aryl-C1-12-alkoxy, Aryl-carbonyl, C1-18 alkoxyalkoxyalkoxyalkoxyaliphatic and hydroxyl, where Het and Aryl are as defined below;
R8 is selected from the group consisting of: hydrogen, nitro, cyano, C1-12 alkoxy, halo, carbonyl-C1-12 alkoxy and halo-C1-12 aliphatic; and Aryl is selected from the group consisting of: phenyl, naphthyl, phenanthryl and anthracenyl;
Cyc is selected from the group consisting of: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl, and optionally has one or more degrees of unsaturation;
Het is a saturated or unsaturated heteroatom ring system selected from the group consisting of: benzimidazole, dihydrothiophene, dioxin, dioxane, dioxolane, dithiane, dithiazine, dithiazole, dithiolane, furan, imidazole, morpholine, oxazole, oxadiazole, oxathiazole, oxathiazolidine, oxazine, oxadiazine, piperazine, piperadine, pyran, pyrazine, pyrazole, pyridine, pyrimidine, pyrrole, pyrrolidine, tetrahydrofuran, tetrazine, thiadiazine, thiadiazole, thiatriazole, thiazine, thiazole, thiomorpholine, thiophene, thiopyran, triazine and triazole;
and the salts, esters, amides, carbamates, solvates, polymorphs, hydrates, affinity reagents and/or prodrugs thereof, in either crystalline or amorphous form. The esters, amides and carbamates are preferably hydrolyzable and are more preferably biohydrolyzeable. The salts are preferably pharmacetically acceptable salts.
A preferred group of compounds of the present invention with respect to the substitutions at R4 are compounds of formula (I):

wherein X is CH;
Y is C or N, with the proviso that when Y is N, R1 is absent, and Z, A and D are each C;
Z is C or N, with the proviso that when Z is N, R2 is absent, and Y, A and D are each C;
A is C or N, with the proviso that when A is N, R3 is absent, and Y, Z and D are each C;
D is C or N, with the proviso that when D is N, Y, Z and A are each C; with the further proviso that Y, Z, A and D do not simultaneously all represent C;
R1 is hydrogen;
R2 is selected from the group consisting of: hydrogen, C1-12 alkoxycarbonyl, Aryl, Het and halogen, where Aryl and Het are as defined below;
R3 is hydrogen or halogen;
R4 is selected from the group consisting of: R7-aminosulfonyl, R7-aminosulfonyl-C1-12 aliphatic, R7-sulfonylamino, R7-sulfonylamino-C1-12 aliphatic, aminosulfonylamino, di-C1-12 aliphatic aminosulfonyl, di-C1-12 aliphatic aminosulfonyl-C1-12 aliphatic, (R8)1-3-Aryl-aminosulfonyl, (R8)1-3-Aryl-sulfonylamino and aminoiminoaminosulfonyl, where R7, R8, Aryl and Het are as defined below;
R5is hydrogen;
R4 and R5 are optionally joined to form a fused ring selected from the group as defined for Het below, and said used ring is optionally substituted by oxo or dioxo;
R7 is selected from the group consisting of: hydrogen, C1-12 aliphatic, C1-12 alkoxy, hydroxy-C1-12 alkoxy, hydroxy-C1-12 aliphatic, carboxylic acid, C1-12 aliphatic-carbonyl, Het, Het-C1-12-aliphatic, Het-C1-12-alkoxy, di-Het-C1-12-alkoxy Aryl, Aryl-C1-12-aliphatic, Aryl-C1-12-alkoxy, Aryl-carbonyl, C1-18 alkoxyalkoxyalkoxyalkoxyaliphatic and hydroxyl, where Het and Aryl are as defined below;
R8 is selected from the group consisting of: hydrogen, nitro, cyano, C1-12 alkoxy, halo, carbonyl-C1-12 alkoxy and halo-C1-12 aliphatic;
Aryl is selected from the group consisting of: phenyl, naphthyl, phenanthryl and anthracenyl;
Cyc is selected from the group consisting of: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl, and optionally has one or more degrees of unsaturation;
Het is a saturated or unsaturated heteroatom ring system selected from the group consisting of: benzimidazole, dihydrothiophene, dioxin, dioxane, dioxolane, dithiane, dithiazine, dithiazole, dithiolane, furan, imidazole, morpholine, oxazole, oxadiazole, oxathiazole, oxathiazolidine, oxazine, oxadiazine, piperazine, piperadine, pyran, pyrazine, pyrazole, pyridine, pyrimidine, pyrrole, pyrrolidine, tetrahydrofuran, tetrazine, thiadiazine, thiadiazole, thiatriazole, thiazine, thiazole, thiomorpholine, thiophene, thiopyran, triazine and triazole;
and the salts, esters, amides, carbamates solvates, polymorphs, hydrates, affinity reagents and/or prodrugs thereof, in either crystalline or amorphous form. The esters, amides and carbamates are preferably hydrolyzable and are more preferably biohydrolyzable. The salts are preferably pharmaceutically acceptable salts.
Due to the presence of an oxindole exocyclic double bond, also included in the compounds of the invention are their respective pure E and Z geometric isomers as well as mixtures of E and Z isomers. The invention as described and claimed does not set any limiting ratios on prevalence of Z to E isomers. Thus, for example, compound number 1 in the tables below is disclosed and claimed as the E geometric isomer thereof, the Z geometric isomer thereof, and a mixture of the E and Z geometric isomers thereof, but not limited by any given ratio(s).
Likewise, it is understood that compounds of formula (I) may exist in tautomeric forms other than that shown in the formula.
Certain of the compounds as described will contain one or more chiral, or asymmetric, centers and will therefore be capable of existing as optical isomers that are either dextrorotatory or levorotatory. Also included in the compounds of the invention are the respective dextrorotatory or levorotatory pure preparations, and mixtures thereof.
Certain compounds of formula (I) above may exist in stereoisomeric forms (e.g. they may contain one or more asymmetric carbon atoms or may exhibit cis-trans isomerism). The individual stereoisomers (enantiomers and diastereoisomers) and mixtures of these are included within the scope of the present invention. Likewise, it is understood that compounds of formula (I) may exist in tautomeric forms other than that shown in the formula, and these are also included within the scope of the present invention.
The present invention also provides compounds of formula (I) and pharmaceutically acceptable salts thereof (hereafter identified as the xe2x80x9cactive compoundsxe2x80x9d) for use in medical therapy, and particularly in the treatment of disorders mediated by CDK2 activity, such as alopecia induced by cancer chemotherapy.
A further aspect of the invention provides a method of treatment of a human or animal body suffering from a disorder kinase is a mitogen activated protein kinase which comprises administering an effective amount of an active compound of formula (I) to the human or animal patient.
Another aspect of the present invention provides the use of an active compound of formula (I), in the preparation of a medicament for the treatment of malignant tumors, or for the treatment of alopecia induced by cancer chemotherapy or induced by radiation therapy. Alternatively, compounds of formula (I) can be used in the preparation of a medicament for the treatment of a disease mediated by a kinase selected from the group consisting of: abl, ARaf, ATK, ATM, bcr-abl, Blk, BRaf, Brk, Btk, CDK1, CDK2, CDK3, CDK4, CDK5, CDK6, CDK7, CDK8, CDK9, cfms, c-fms, c-kit, c-met, cRaf1, CSF1R, CSK, c-src, EGFR, ErbB2, ErbB3, ErbB4, ERK, ERK1, ERK2, Fak, fes, FGFR1, FGFR2, FGFR3, FGFR4, FGFR5, Fgr, FLK-4, Fps, Frk, Fyn, GSK, gsk3a, gsk3b, Hck, IGF-1R, IKK, IKK1, IKK2, IKK3, INS-R, Integrin-linkedkinase, Jak, JAK1, JAK2, JAK3, JNK, JNK, Lck, Lyn, MEK, MEK1, MEK2, p38, PDGFR, PIK, PKB1, PKB2, PKB3, PKC, PKCxcex1, PKCxcex2, PKCxcex4, PKCxcex5, PKCxcex3, PKCxcex, PKCxcexc, PKCxcex6, PLK1, Polo-like kinase, PYK2, tie1, tie2, TrkA, TrkB, TrkC, UL13, UL97, VEGF-R1, VEGF-R2, Yes and Zap70. Additionally, compounds of formula (I) can be used in the preparation of a medicament for the treatment of a disease or disorder such as organ transplant rejection, tumor growth, chemotherapy-induced alopecia, chemotherapy-induced thrombocytopenia, chemotherapy-induced leukopenia, mucocitis, plantar-palmar syndrome, restenosis, atherosclerosis, rheumatoid arthritis, angiogenesis, hepatic cirrhosis, glomerulonephritis, diabetic nephropathy, malignant nephrosclerosis, thrombotic microangiopathy, glomerulopathy, psoriasis, diabetes mellitus, inflammation, neurodegenerative disease, macular. degeneration, actinic keratosis and hyperproliferative disorders.
Another aspect of the present invention provides the use of an active compound of formula (I), in coadministration with previously known anti-tumor therapies for more effective treatment of such tumors.
Another aspect of the present invention provides the use of an active compound of formula (I) in the preparation of a medicament for the treatment of viral or eukaryotic infection.
Other aspects of the present invention related to the inhibition of mitogen activated protein kinases are discussed in more detail below.
Compounds we have synthesized as part of the present invention which are currently preferred are listed in Tables 1 and 2 below. Compounds are identified by the numbers shown in the first column; variables below in the rest of the columns are with reference to the generic structure (I). Corresponding IUPAC nomenclature are disclosed in Table 2. Since all substituents at each point of substitution are capable of independent synthesis of each other, the tables are to be read as a matrix in which any combination of substituents is within the scope of the disclosure and claims of the invention.
Standard accepted nomenclature corresponding to the Examples set forth in this specification are set forth below. In some cases nomenclature is given for one or more possible isomers.
The invention discloses 10 different points of substitution on structural foirmula (I). Each of these points of substitution bears a substituent whose selection and synthesis as part of this invention was independent of all other points of substitution on formula (I). Thus, each point of substitution is now further described individually.
Preferred substitutions at the R1 position include hydrogen, halogen, amide, nitro, lower alkyl, hydroxy, hydroxyalkyl, pyrimidineloweralkyl, loweralkoxycarbonyl, cyclic loweralkyl, hydroxyphenylloweralkyl, phenoxy, aloxy and pyrazole; and R1 fused with R2to form a fused ring selected from the group consisting of: thiazole, pyrazole, triazole, halogen-substituted diazole, acyl substituted pyrrole and pyridine. Most preferred substituents at R1 include hydrogen and methyl and R1 fused with R2 for form fused thiazole or fused pyridine. The most highly preferred substitution at the R1 position is hydrogen.
Preferred substitutions at the R2 position include hydrogen, halogen, sulfate, amine, quatemary amine, amide, ester, phenyl, alkoxy, aminosulfonyl, lower alkyl sulfonyl, furanyl lower alkyl amide, pyridinyl lower alkyl amide, alkoxy-substituted phenyl lower alkyl amide, morpholino lower alkyl amide, imidazolyl lower alkyl amide, hydroxy lower alkyl amide, alkoxy lower alkyl amide, lower alkyl amide, lower alkyl sulfonamide, lower alkyl hydroxy substituted amino, nitro, halogen-substituted phenoxycarbonyl and triazole and oxazole rings, or are R2 fused with R3 to form a fused ring selected from the group consisting of: oxazole, pyrrole, and dioxolane, which fused ring is optionally substituted by lower alkyl or lower alkyl carbonyl, and which fused ring is optionally a hetero ring having nitrogen as the heteroatom and forming a quatemary ammonium salt ionically bonded with a halogen atom. Most preferred substituents at R2 include hydrogen, phenyl, 2-furanyl, 3-thiophenyl, bromo and carbethoxy.
Preferred substitutions at R3 include hydrogen, lower alkyl, hydroxy lower alkyl, halogen, phenoxy and alkoxy. Most preferred include hydrogen and chloro. Most highly preferred is hydrogen.
Preferred substitutions at R4 include sulfonylamino, sulfonylaminoamino, lower alkyl sulfonylamino, lower alkylsulfonyl lower alkyl, alkoxysulfonylamino, phenylcarbonylsulfonylamino, phenoxysulfonyl, hydroxy lower alkylsulfonylamino, hydroxy lower alkylsulfonylamino lower alkyl, alkyl, phenylsulfonylamino (optionally substituted by halogensubstituted lower alkyl), aminoiminosulfonylamino, alkylsulfonylaminoalkyl, pyridinyl lower alkyl sulfonylamino, benzamideazolesulfonylamino, pyridylsulfonylamino, pyrimidinylsulfonylamino, thiadiazolylsulfonylamino (optionally substituted by lower alkyl), thiazolesulfonylamino, hydroxyalkoxyalkylsulfonylamino and 4xe2x80x2-SO2NH[(CH2)2O]4CH3, or R4 fused with R5to form a fused ring selected from the group consisting of imidazole, triazole, cyclic sulfonylamino and thiaphene, where said fused ring is optionally disubstituted on the sulfur heteroatom by oxo. The most preferred substitutions include 2-pyridine sulfonylamino, 4-pyridine sulfonylamino, hydroxy n-butyl sulfonylamino, methylsulfonylaminomethylene, sulfonyldimethylamino, fused 1,2-pyrazole and sulfonylamino. In a most highly preferred embodiment, R4 is sulfonylamino or fused 1,2-pyrazole.
The preferred substitution at R5 is hydrogen.
Preferred substitutions at X include N, CH and CCH3. Most preferred is CH.
The preferred substitution at Y is N or C.
The preferred substitution at Z is N or C.
The preferred substitution at A is N or C.
The preferred substitution at D is N or C.
Salts encompassed within the term xe2x80x9cpharmaceutically acceptable saltsxe2x80x9d refer to non-toxic salts of the compounds of this invention which are generally prepared by reacting the free base with a suitable organic or inorganic acid or by reacting the acid with a suitable organic or inorganic base. Representative salts include the following salts: Acetate, Benzenesulfonate, Benzoate, Bicarbonate, Bisulfate, Bitartrate, Borate, Bromide, Calcium Edetate, Camsylate, Carbonate, Chloride, Clavulanate, Citrate, Diethanolamine, Dihydrochloride, Edetate, Edisylate, Estolate, Esylate, Fumarate, Gluceptate, Gluconate, Glutamate, Glycollylarsanilate, Hexylresorcinate, Hydrabamine, Hydrobromide, Hydrocloride, Hydroxynaphthoate, Iodide, Isethionate, Lactate, Lactobionate, Laurate, Malate, Maleate, Mandelate, Mesylate, Metaphosphoric, Methylbromide, Methyinitrate, Methylsulfate, Monopotassium Maleate, Mucate, Napsylate, Nitrate, N-methylglucamine, Oxalate, Pamoate (Embonate), Palmitate, Pantothenate, Phosphate/diphosphate, Polygalacturonate, Potassium, Salicylate, Sodium, Stearate, Subacetate, Succinate, Tannate, Tartrate, Teoclate, Tosylate, Trifluoroacetate, Triethiodide, Trimethylammonium and Valerate.
Other salts which are not pharmaceutically acceptable may be useful in the preparation of compounds of formula (I) and these form a further aspect of the invention.
Also included within the scope of the invention are the individual isomers of the compounds represented by formula (I) above as well as any wholly or partially equilibrated mixtures thereof. The present invention also covers the individual isomers of the compounds represented by formula above as mixtures with isomers thereof in which one or more chiral asymmetric centers are inverted.
As used herein, the term xe2x80x9caliphaticxe2x80x9d refers to the terms alkyl, alkylene, alkenyl, alkenylene, alkynyl and alkynylene.
As used herein, the term xe2x80x9clowerxe2x80x9d refers to a group having between one and six carbons.
As used herein, the term xe2x80x9calkylxe2x80x9d refers to a straight or branched chain hydrocarbon having from one to twelve carbon atoms, optionally substituted with substituents selected from the group consisting of: lower alkyl, lower alkoxy, lower alkylsulfanyl, lower alkylsulfenyl, lower alkylsulfonyl, oxo, hydroxy, mercapto, amino optionally substituted by alkyl, carboxy, carbamoyl optionally substituted by alkyl, aminosulfonyl optionally substituted by alkyl, nitro, cyano, halogen, or lower perfluoroalkyl, multiple degrees of substitution being allowed. Examples of xe2x80x9calkylxe2x80x9d as used herein include, but are not limited to, n-butyl, n-pentyl, isobutyl and isopropyl, and the like.
As used herein, the term xe2x80x9calkylenexe2x80x9d refers to a straight or branched chain divalent hydrocarbon radical having from one to ten carbon atoms, optionally substituted with substituents selected from the group consisting of: lower alkyl, lower alkoxy, lower alkylsulfanyl, lower alkylsulfenyl, lower alkylsulfonyl, oxo, hydroxy, mercapto, amino optionally substituted by alkyl, carboxy, carbamoyl optionally substituted by alkyl, aminosulfonyl optionally substituted by alkyl, nitro, cyano, halogen, or lower perfluoroalkyl, multiple degrees of substitution being allowed. Examples of xe2x80x9calkylenexe2x80x9d as used herein include, but are not limited to, methylene, ethylene, and the like.
As used herein, the term xe2x80x9calkenylxe2x80x9d refers to a hydrocarbon radical having from two to ten carbons and at least one carbonxe2x80x94carbon double bond, optionally substituted with substituents selected from the group consisting of: lower alkyl, lower alkoxy, lower alkylsulfanyl, lower alkylsulfenyl, lower alkylsulfonyl, oxo, hydroxy, mercapto, amino optionally substituted by alkyl, carboxy, carbamoyl optionally substituted by alkyl, aminosulfonyl optionally substituted by alkyl, nitro, cyano, halogen, or lower perfluoroalkyl, multiple degrees of substitution being allowed.
As used herein, the term xe2x80x9calkenylenexe2x80x9d refers to an straight or branched chain divalent hydrocarbon radical having from two to ten carbon atoms and one or more carbonxe2x80x94carbon double bonds, optionally substituted with substituents selected from the group consisting of: lower alkyl, lower alkoxy, lower alkylsulfanyl, lower alkylsulfenyl, lower alkylsulfonyl, oxo, hydroxy, mercapto, amino optionally substituted by alkyl, carboxy, carbamoyl optionally substituted by alkyl, aminosulfonyl optionally substituted by alkyl, nitro, cyano, halogen, or lower perfluoroalkyl, multiple degrees of substitution being allowed. Examples of xe2x80x9calkenylenexe2x80x9d as used herein include, but are not limited to, ethene-1,2-diyl, propene-1,3-diyl, methylene-1,1-diyl, and the like.
As used herein, the term xe2x80x9calkynylxe2x80x9d refers to a hydrocarbon radical having from two to ten carbons and at least one carbonxe2x80x94carbon triple bond, optionally substituted with substituents selected from the group consisting of: lower alkyl, lower alkoxy, lower alkylsulfanyl, lower alkylsulfenyl, lower alkylsulfonyl, oxo, hydroxy, mercapto, amino optionally substituted by alkyl, carboxy, carbamoyl optionally substituted by alkyl, aminosulfonyl optionally substituted by alkyl, nitro, cyano, halogen, or lower perfluoroalkyl, multiple degrees of substitution being allowed.
As used herein, the term xe2x80x9calkynylenexe2x80x9d refers to a straight or branched chain divalent hydrocarbon radical having from two to ten carbon atoms and one or more carbonxe2x80x94carbon triple bonds, optionally substituted with substituents selected from the group consisting of: lower alkyl, lower alkoxy, lower alkylsulfanyl, lower alkylsulfenyl, lower alkylsulfonyl, oxo, hydroxy, mercapto, amino optionally substituted by alkyl, carboxy, carbamoyl optionally substituted by alkyl, aminosulfonyl optionally substituted by alkyl, nitro, cyano, halogen, or lower perfluoroalkyl, multiple degrees of substitution being allowed. Examples of xe2x80x9calkynylenexe2x80x9d as used herein include, but are not limited to, ethyne-1,2-diyl, propyne-1,3-diyl, and the like.
As used herein, the term xe2x80x9ccycloaliphaticxe2x80x9d refers to the terms cycloalkyl, cycloalkylene, cycloalkenyl, cycloalkenylene, cycloalkynyl and cycloalkylnylene.
As used herein, xe2x80x9ccycloalkylxe2x80x9d refers to a alicyclic hydrocarbon group with one or more degrees of unsaturation, having from three to twelve carton atoms, optionally substituted with substituents selected from the group consisting of: lower alkyl, lower alkoxy, lower alkylsulfanyl, lower alkylsulfenyl, lower alkylsulfonyl, oxo, hydroxy, mercapto, amino optionally substituted by alkyl, carboxy, carbamoyl optionally substituted by alkyl, aminosulfonyl optionally substituted by alkyl, nitro, cyano, halogen, or lower perfluoroalkyl, multiple degrees of substitution being allowed. xe2x80x9cCycloalkylxe2x80x9d includes, by way of example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, or cyclooctyl, and the like.
As used herein, the term xe2x80x9ccycloalkylenexe2x80x9d refers to an non-aromatic alicyclic divalent hydrocarbon radical having from three to twelve carbon atoms, optionally substituted with substituents selected from the group consisting of: lower alkyl, lower alkoxy, lower alkylsulfanyl, lower alkylsulfenyl, lower alkylsulfonyl, oxo, hydroxy, mercapto, amino optionally substituted by alkyl, carboxy, carbamoyl optionally substituted by alkyl, aminosulfonyl optionally substituted by alkyl, nitro, cyano, halogen, or lower perfluoroalkyl, multiple degrees of substitution being allowed. Examples of xe2x80x9ccycloalkylenexe2x80x9d as used herein include, but are not limited to, cyclopropyl-1,1-diyl, cyclopropyl-1,2-diyl, cyclobutyl-1,2-diyl, cyclopentyl-1,3-diyl, cyclohexyl-1,4-diyl, cycloheptyl-1,4-diyl, or cyclooctyl-1,5-diyl, and the like.
As used herein, the term xe2x80x9ccycloalkenylxe2x80x9d refers to a substituted alicyclic hydrocarbon radical having from three to twelve carbon atoms and at least one carbon-carbon double bond in the ring system, optionally substituted with substituents selected from the group consisting of: lower alkyl, lower alkoxy, lower alkylsulfanyl, lower alkylsulfenyl, lower alkylsulfonyl, oxo, hydroxy, mercapto, amino optionally substituted by alkyl, carboxy, carbamoyl optionally substituted by alkyl, aminosulfonyl optionally substituted by alkyl, nitro, cyano, halogen, or lower perfluoroalkyl, multiple degrees of substitution being allowed. Examples of xe2x80x9ccycloalkenylenexe2x80x9d as used herein include, but are not limited to, 1-cyclopentene-3-yl, 1-cyclohexene-3-yl, 1-cycloheptene-4-yl, and the like.
As used herein, the term xe2x80x9ccycloalkenylenexe2x80x9d refers to a substituted alicyclic divalent hydrocarbon radical having from three to twelve carbon atoms and at least one carbon-carbon double bond in the ring system, optionally substituted with substituents selected from the group consisting of: lower alkyl, lower alkoxy, lower alkylsulfanyl, lower alkylsulfenyl, lower alkylsulfonyl, oxo, hydroxy, mercapto, amino optionally substituted by alkyl, carboxy, carbamoyl optionally substituted by alkyl, aminosulfonyl optionally substituted by alkyl, nitro, cyano, halogen, or lower perfluoroalkyl, multiple degrees of substitution being allowed. Examples of xe2x80x9ccycloalkenylenexe2x80x9d as used herein include, but are not limited to, 4,5-cyclopentene-1,3-diyl, 3,4-cyclohexene-1,1-diyl, and the like.
As used herein, the term xe2x80x9cheteroatom ring systemxe2x80x9d is inclusive of heterocyclic, heterocyclyl, heteroaryl and heteroarylene ring systems. Non-limiting examples of such heteroatom ring systems are recited in the Summary of the Invention, above.
As used herein, the term xe2x80x9cheterocyclicxe2x80x9d or the term xe2x80x9cheterocyclylxe2x80x9d refers to a three to twelve-membered heterocyclic ring having one or more degrees of unsaturation containing one or more heteroatomic substitutions selected from S, SO, SO2, O, or N, optionally substituted with substituents selected from the group consisting of: lower alkyl, lower alkoxy, lower alkylsulfanyl, lower alkylsulfenyl, lower alkylsulfonyl, oxo, hydroxy, mercapto, amino optionally substituted by alkyl, carboxy, carbamoyl optionally substituted by alkyl, aminosulfonyl optionally substituted by alkyl, nitro, cyano, halogen, or lower perfluoroalkyl, multiple degrees of substitution being allowed. Such a ring may be optionally fused to one or more of another xe2x80x9cheterocyclicxe2x80x9d ring(s) or cycloalkyl ring(s). Examples of xe2x80x9cheterocyclicxe2x80x9d include, but are not limited to, tetrahydrofuran, pyran, 1,4-dioxane, 1,3-dioxane, piperidine, pyrrolidine, morpholine, tetrahydrothiopyran, tetrahydrothiophene, and the like.
As used herein, the term xe2x80x9cheterocyclylenexe2x80x9d refers to a three to twelve-membered heterocyclic ring diradical having one or more degrees of unsaturation containing one or more heteroatoms selected from S, SO, SO2, O, or N, optionally substituted with substituents selected from the group consisting of: lower alkyl, lower alkoxy, lower alkylsulfanyl, lower alkylsulfenyl, lower alkylsulfonyl, oxo, hydroxy, mercapto, amino optionally substituted by alkyl, carboxy, carbamoyl optionally substituted by alkyl, aminosulfonyl optionally substituted by alkyl, nitro, cyano, halogen, or lower perfluoroalkyl, multiple degrees of substitution being allowed. Such a ring may be optionally fused to one or more benzene rings or to one or more other xe2x80x9cheterocyclicxe2x80x9d rings or cycloalkyl rings. Examples of xe2x80x9cheterocyclylenexe2x80x9d rings include, but are not limited to, tetrahydrofuran-2,5-diyl, morpholine-2,3-diyl, pyran-2,4-diyl, 1,4-dioxane-2,3-diyl, 1,3-dioxane-2,4-diyl, piperidine-2,4-diyl, piperidine-1,4-diyl, pyrrolidine-1,3-diyl, morpholine-2,4-diyl, and the like.
As used herein, the term xe2x80x9carylxe2x80x9d refers to a benzene ring or to an optionally substituted benzene ring system fused to one or more optionally substituted benzene rings to form anthracene, phenanthrene, or napthalene ring systems, optionally substituted with substituents selected from the group consisting of: lower alkyl, lower alkoxy, lower alkylsulfanyl, lower alkylsulfenyl, lower alkylsulfonyl, oxo, hydroxy, mercapto, amino optionally substituted by alkyl, carboxy, tetrazolyl, carbamoyl optionally substituted by alkyl, aminosulfonyl optionally substituted by alkyl, acyl, aroyl, heteroaroyl, acyloxy, aroyloxy, heteroaroyloxy, alkoxycarbonyl, nitro, cyano, halogen, lower perfluoroalkyl, heteroaryl, or aryl, multiple degrees of substitution being allowed. Examples of aryl include, but are not limited to, phenyl, 2-naphthyl, 1-naphthyl, biphenyl, and the like.
As used herein, the term xe2x80x9carylenexe2x80x9d refers to a benzene ring diradical or to a benzene ring system diradical fused to one or more optionally substituted benzene rings, optionally substituted with substituents selected from the group consisting of: lower alkyl, lower alkoxy, lower alkylsulfanyl, lower alkylsulfenyl, lower alkylsulfonyl, oxo, hydroxy, mercapto, amino optionally substituted by alkyl, carboxy, tetrazolyl, carbamoyl optionally substituted by alkyl, aminosulfonyl optionally substituted by alkyl, acyl, aroyl, heteroaroyl, acyloxy, aroyloxy, heteroaroyloxy, alkoxycarbonyl, nitro, cyano, halogen, lower perfluoroalkyl, heteroaryl, or aryl, multiple degrees of substitution being allowed. Examples of xe2x80x9carylenexe2x80x9d include, but are not limited to, benzene-1,4-diyl, naphthalene-1,8-diyl, anthracene-1,4-diyl, and the like.
As used herein, the term xe2x80x9cheteroarylxe2x80x9d refers to a fivexe2x80x94to sevenxe2x80x94membered aromatic ring, or to a polycyclic heterocyclic aromatic ring, containing one or more nitrogen, oxygen, or sulfur heteroatoms at any position, where N-oxides and sulfur monoxides and sulfur dioxides are permissible heteroaromatic substitutions, optionally substituted with substituents selected from the group which includes lower alkyl, lower alkoxy, lower alkylsulfanyl, lower alkylsulfenyl, lower alkylsulfonyl, oxo, hydroxy, mercapto, amino optionally substituted by alkyl, carboxy, tetrazolyl, carbamoyl optionally substituted by alkyl, aminosulfonyl optionally substituted by alkyl, acyl, aroyl, heteroaroyl, acyloxy, aroyloxy, heteroaroyloxy, alkoxycarbonyl, nitro, cyano, halogen, lower perfluoroalkyl, heteroaryl and aryl, multiple degrees of substitution being allowed. For polycyclic aromatic ring systems, one or more of the rings may contain one or more heteroatoms. Examples of xe2x80x9cheteroarylxe2x80x9d used herein are furan, thiophene, pyrrole, imidazole, pyrazole, triazole, tetrazole, thiazole, oxazole, isoxazole, oxadiazole, thiadiazole, isothiazole, pyridine, pyridazine, pyrazine, pyrimidine, quinoline, isoquinoline, benzofuran, benzothiophene, indole and indazole, and the like.
As used herein, the term xe2x80x9cheteroarylenexe2x80x9d refers to a fivexe2x80x94to sevenxe2x80x94membered aromatic ring diradical, or to a polycyclic heterocyclic aromatic ring diradical, containing one or more nitrogen, oxygen, or sulfur heteroatoms, where N-oxides and sulfur monoxides and sulfur dioxides are permissible heteroaromatic substitutions, optionally substituted with substituents selected from the group consisting of: lower alkyl, lower alkoxy, lower alkylsulfanyl, lower alkylsulfenyl, lower alkylsulfonyl, oxo, hydroxy, mercapto, amino optionally substituted by alkyl, carboxy, tetrazolyl, carbamoyl optionally substituted by alkyl, aminosulfonyl optionally substituted by alkyl, acyl, aroyl, heteroaroyl, acyloxy, aroyloxy, heteroaroyloxy, alkoxycarbonyl, nitro, cyano, halogen, lower perfluoroalkyl, heteroaryl, or aryl, multiple degrees of substitution being allowed. For polycyclic aromatic ring system diradicals, one or more of the rings may contain one or more heteroatoms. Examples of xe2x80x9cheteroarylenexe2x80x9d used herein are furan-2,5-diyl, thiophene-2,4-diyl, 1,3,4-oxadiazole-2,5-diyl, 1,3,4-thiadiazole-2,5-diyl, 1,3-thiazole-2,4-diyl, 1,3-thiazole-2,5-diyl, pyridine-2,4-diyl, pyridine-2,3-diyl, pyridine-2,5-diyl, pyrimidine-2,4-diyl, quinoline-2,3-diyl, and the like.
As used herein, the term xe2x80x9calkoxyxe2x80x9d refers to the group RaOxe2x80x94, where Ra is aliphatic.
As used herein, the term xe2x80x9calkylsulfanylxe2x80x9d refers to the group RaSxe2x80x94, where Ra is aliphatic.
As used herein, the term xe2x80x9calkylsulfenylxe2x80x9d refers to the group RaS(O)xe2x80x94, where Ra is aliphatic.
As used herein, the termr xe2x80x9calkylsulfonylxe2x80x9d refers to the group RaSO2xe2x80x94, where Ra is aliphatic.
As used herein, the term xe2x80x9cacylxe2x80x9d refers to the group RaC(O)xe2x80x94, where Ra is aliphatic, cycloaliphatic, or heterocyclyl.
As used herein, the term xe2x80x9caroylxe2x80x9d refers to the group RaC(O)xe2x80x94, where Ra is aryl.
As used herein, the term xe2x80x9cheteroaroylxe2x80x9d refers to the group RaC(O)xe2x80x94, where Ra is heteroaryl.
As used herein, the term xe2x80x9calkoxycarbonylxe2x80x9d refers to the group RaOC(O)xe2x80x94, where Ra is aliphatic.
As used herein, the term xe2x80x9cacyloxyxe2x80x9d refers to the group RaC(O)Oxe2x80x94, where Ra is aliphatic, cycloaliphatic, or heterocyclyl.
As used herein, the term xe2x80x9caroyloxyxe2x80x9d refers to the group RaC(O)Oxe2x80x94, where Ra is aryl.
As used herein, the term xe2x80x9cheteroaroyloxyxe2x80x9d refers to the group RaC(O)Oxe2x80x94, where Ra is heteroaryl.
As used herein, the term xe2x80x9coptionallyxe2x80x9d is inclusive of embodiments in which a described condition is present and embodiments in which such described condition is not present, for example, where the term is used with reference to a chemical substituent, it indicates the inclusion of embodiments in which the specified substituent is present as well as embodiments in which the specified substituent is not present.
As used herein, the term xe2x80x9csubstitutedxe2x80x9d refers to substitution with the named substituent or substituents, multiple degrees of substitution being allowed.
As used herein, the terms xe2x80x9ccontainxe2x80x9d or xe2x80x9ccontainingxe2x80x9d in reference to any of the above-defined alkyl, alkenyl, alkynyl or cycloalkyl substituents, are inclusive of in-line substitutions at any position along such alkyl, alkenyl, alkynyl or cycloalkyl substituents, with one or more of any of O, S, SO, SO2, N, or N-alkyl, including, for example; xe2x80x94CH2xe2x80x94Oxe2x80x94CH2xe2x80x94, xe2x80x94CH2xe2x80x94SO2xe2x80x94CH2xe2x80x94, xe2x80x94CH2xe2x80x94NHxe2x80x94CH3 and so forth.
As used herein, the term xe2x80x9csolvatexe2x80x9d is a complex of variable stoichiometry formed by a solute (in this invention, a compound of formula (I)) and a solvent. Such solvents for the purpose of the invention may not interfere with the biological activity of the solute. Solvents may be, by way of example, water, ethanol, or acetic acid.
The compounds of the present invention have the ability to crystallize in more than one form, a characteristic which is known as polymorphism, and such polymorphic forms (xe2x80x9cpolymorphsxe2x80x9d) are within the scope of the present invention. Polymorphism generally can occur as a response to changes in temperature or pressure or both and can also result from variations in the crystallization process. Polymorphs can be distinguished by various physical characteristics known in the art such as x-ray diffraction patterns, solubility, and melting point.
As used herein, the terms xe2x80x9cbiohydrolyzable carbonatexe2x80x9d, xe2x80x9cbiohydrolyzable ureidexe2x80x9d and xe2x80x9cbiohydrolyzable carbamatexe2x80x9d include carbonates, ureides, and carbamates, respectively, of a compound of the general formula (I) which carbonates, ureides, and carbamates, do not completely diminish the biological activity of the parent substance. Such biohydrolyzable carbonates, ureides, and carbamates may confer on the parent compound of the general formula (I) advantageous properties in vivo, such as improved duration of action, onset of action, and the like. Also included are compounds which are relatively biologically inactive but which are converted in vivo by the subject to the biologically active principle. An advantage of such biohydrolyzable forms is that, for example, they facilitate improved oral administration because the carbonates, ureides, and carbamates are more readily absorbed from the gut and are then transformed to a compound of formula (I) in plasma. Many of such biohydrolyzable compounds are known in the art and include, by way of example, lower alkyl carbamates.
As used herein, the term xe2x80x9cbiohydrolyzable esterxe2x80x9d is an ester of a compound of general formula which does not completely diminish the biological activity of the parent substance. Such esters may confer on the parent compound of the general formula (I) advantageous properties in vivo, such as improved duration of action, onset of action, and the like. Also included are esters which are relatively biologically inactive but which are converted in vivo by the subject to the biologically active principle. An advantage of such biohydrolyzable esters is that, for example, they facilitate improved oral administration because they are more readily absorbed from the gut and are then transformed to a compound of formula (I) in plasma. Many biohydrolyzable esters are known in the art and include, by way of example, lower alkyl esters, lower acyloxy-alkyl esters, lower alkoxyacyloxyalkyl esters, alkoxyacyloxy esters, alkyl acylamino alkyl esters and choline esters.
As used herein, the term xe2x80x9cbiohydrolyzable amidexe2x80x9d is an amide of a compound of general formula which does not completely diminish the biological activity of the parent substance. Such amides may confer on the parent compound of the general formula (I) advantageous properties in vivo, such as improved duration of action, onset of action, and the like. Also included are amides which are relatively biologically inactive but which are converted in vivo by the subject to the biologically active principle. An advantage of such biohydrolyzable amides is that, for example, they facilitate improved oral administration because they are more readily absorbed from the gut and are then transformed to a compound of formula (I) in plasma. Many biohydrolyzable amides are known in the art and include, by way of example, lower alkyl amides, xcex1-amino acid amides, alkoxyacyl amides and alkylaminoalkylcarbonyl amides.
As used herein, the term xe2x80x9cprodrugxe2x80x9d includes biohydrolyzable amides, biohydrolyzable esters and biohydrolyzable carbamates and also encompasses compounds in which the biohydrolyzable functionality in such prodrug is encompassed in the compound of formula (I): for example, a lactam formed by a carboxylic group in R1 and an amine in R2, and compounds which may be oxidized or reduced biologically at a given functional group to yield drug substances of formula (I). Examples of these functional groups are, but are not limited to, 1,4-dihydropyridine, N-alkylcarbonyl-1,4-dihydropyridine, 1,4-cyclohexadiene, tert-butyl, and the like.
As used herein, the term xe2x80x9caffinity reagentxe2x80x9d is a group attached to the compound of formula (I) which does not affect its in vitro biological activity, allowing the compound to bind to a target, yet such a group binds strongly to a third component allowing (a) characterization of the target as to localization within a cell or other organism component, perhaps by visualization by fluorescence or radiography, or (b) facile separation of the target from an unknown mixture of targets, whether proteinaceous or not proteinaceous. An example of an affinity reagent according to (b) would be biotin either directly attached to (I) or linked with a spacer of one to 50 atoms selected from the group consisting of C, H, O, N, S, or P in any combination. An example of an affinity reagent according to (a) above would be fluorescein, either directly attached to (I) or linked with a spacer of one to 50 atoms selected from the group consisting of C, H, O, N, S, or P in any combination.
The term xe2x80x9ceffective amountxe2x80x9d means that amount of a drug or pharmaceutical agent that will elicit the biological or medical response of a tissue, system, animal or human that is being sought by a researcher or clinician. The term xe2x80x9ctherapeutically effective amountxe2x80x9d means any amount which, as compared to a corresponding subject who has not received such amount, results in improved treatment, healing, prevention, or amelioration of a disease or disorder, or a decrease in the rate of advancement of a disease or disorder, and also includes amounts effective to enhance normal physiological function.
Whenever the terms xe2x80x9caliphaticxe2x80x9d or xe2x80x9carylxe2x80x9d or either of their prefixes appear in a name of a substituent (e.g. arylalkoxyaryloxy) they shall be interpreted as including those limitations given above for xe2x80x9caliphaticxe2x80x9d and xe2x80x9carylxe2x80x9d. Aliphatic or cycloalkyl substituents shall be recognized as being term equivalents to those having one or more degrees of unsaturation. Designated numbers of carbon atoms (e.g. C1-10) shall refer independently to the number of carbon atoms in an aliphatic or cyclic aliphatic moiety or to the aliphatic portion of a larger substituent in which the term xe2x80x9caliphaticxe2x80x9d appears as a prefix (e.g. xe2x80x9cal-xe2x80x9d).
As used herein, the term xe2x80x9cdisubstituted aminexe2x80x9d or xe2x80x9cdisubstituted amino-xe2x80x9d shall be interpreted to include either one or two substitutions on that particular nitrogen atom.
As used herein, the term xe2x80x9coxoxe2x80x9d shall refer to the substituent xe2x95x90O.
As used herein, the term xe2x80x9chalogenxe2x80x9d or xe2x80x9chaloxe2x80x9d shall include iodine, bromine, chlorine and fluorine.
As used herein, the term xe2x80x9cmercaptoxe2x80x9d shall refer to the substituent xe2x80x94SH.
As used herein, the term xe2x80x9ccarboxyxe2x80x9d shall refer to the substituent xe2x80x94COOH.
As used herein, the term xe2x80x9ccyanoxe2x80x9d shall refer to the substituent xe2x80x94CN.
As used herein, the term xe2x80x9caminosulfonylxe2x80x9d shall refer to the substituent xe2x80x94SO2NH2.
As used herein, the term xe2x80x9ccarbamoylxe2x80x9d shall refer to the substituent xe2x80x94C(O)NH2.
As used herein, the term xe2x80x9csulfanylxe2x80x9d shall refer to the substituent xe2x80x94Sxe2x80x94.
As used herein, the term xe2x80x9csulfenylxe2x80x9d shall refer to the substituent xe2x80x94S(O)xe2x80x94.
As used herein, the term xe2x80x9csulfonylxe2x80x9d shall refer to the substituent xe2x80x94S(O)2xe2x80x94.
The compounds of formula (I) are readily synthesized using various synthetic procedures known in the art and are readily prepared according to the following reaction Synthesis Schemes (in which all variables are as defined herein) and examples or modifications thereof using readily available starting materials, reagents and conventional synthesis procedures. In these reactions, it is also possible to make use of variants which are themselves known to those of ordinary skill in this art, but are not mentioned in greater detail.





In the foregoing Schemes 1-5, R is R4 and/or R5 as described herein.

wherein R is Het, Ar or CO2Et, where Het and Ar are as described herein.
The following are three preferred synthetic schemes according to the present invention:

The most preferred compounds of the invention are any or all of those specifically set forth in these examples. These compounds are not, however, to be construed as forming the only genus that is considered as the invention, and any combination of the compounds or their moieties may itself form a genus. The following examples further illustrate details for the preparation of the compounds of the present invention. Those skilled in the art will readily understand that known variations of the conditions and processes of the following preparative procedures can be used to prepare these compounds. All temperatures are degrees Celsius unless noted otherwise.
Abbreviations used in the Examples are as follows:
Reagents are commercially available or are prepared according to procedures in the literature. The physical data given for the compounds exemplified is consistent with the assigned structure of those compounds. 1H NMR spectra were obtained on VARIAN Unity Plus NMR spectrophotometers at 300 or 400 Mhz. Mass spectra were obtained on Micromass Plafform II mass spectrometers from Micromass Ltd. Altrincham, UK, using either Atmospheric Chemical Ionization (APCI) or Electrospray Ionization (ESI). Analytical thin layer chromatography (TLC) was used to verify the purity of some intermediates which could not be isolated or which were too unstable for full characterisation, and to follow the progess of reactions. Unless otherwise stated, this was done using silica gel (Merck Silica Gel 60 F254). Unless otherwise stated, column chromatography for the purification of some compounds, used Merck Silica gel 60 (230-400 mesh), and the stated solvent system under pressure.
A Micromass Platform II mass spectrometer equipped with an electrospray ion source was used to acquire low resolution LC-MS data for the samples that were prepared in library format. The system software runs on a PC computer with the Microsoft operating system, and consists of Masslynx v3.1 and Openlynx v3.1 software packages. The mass spectrometer inlet system was comprised of a Hewlett Packard 1100 HPLC Chromatograph, a Gilson 215 autosampler, and a Hewlett Packard 1100 photodiode array detector. A Supelco ABZ+5 cm column was used to provide separations prior to electrospray ionization. The HPLC was programmed as follows:
The data were processed automatically using standard peak detection parameters provided by the Openlynx software.
Micromass LCT bench-top mass spectrometer equipped with an electrospray ionization source was used to obtain accurate mass data for the samples that were prepared in library format. The LCT utilizes two hexapole RF lenses to transfer ions from the source to an orthogonal acceleration time-of-flight (TOF) analyser. The ions emerging from the analyser are detected using a dual microchannel plate detector and ion counting system. The system software runs on a PC computer with the Microsoft operating system, and consists of Masslynx v3.2 and Openlynx v3.2 software packages. The mass spectrometer inlet system is comprised of a Waters Alliance 2690 Separations Module, Waters 2700 autosampler, Waters 996 photo-diode array detector and Valco column switching device. A mobile phase flow rate of 1 ml/min exits the Alliance 2690 and is reduced to a mass spectrometer flow rate of 20 ul/min using an Acurate flow splitter. A lock mass solution at a flow rate of 4 ul/min is added to the spectrometer flow via a Harvard syringe pump and a tee piece placed immediately before the electrospray probe. The instrument resolution was determined by acquiring a spectrum and measuring the full peak width t half peak height (FWHH). The instrument was tuned to provide a resolution of 4600 to 5000 (FWHH). The instrument was calibrated using the ions of polyethylene glycol (PEG) as reference standards. The lock mass used [3,5-Dil-Tyr, Ala,N-Me-Phe, Gly-0l] Enkephalin (MH+C26H34I2N5O6=766.0599) at a concentration of 5 ng/ul.